A set of plasmids conferring resistance to several antibiotics, including the combination of trimethoprim and sulfamethoxazole, has been isolated from Escherichia coli following conjugative cotransfer from a clinical isolate of Shigella flexneri 2a. One of the plasmids, pCN1, was shown by subcloning and DNA sequencing to carry a gene encoding a trimethoprim-insensitive dihydrofolate reductase identical to that found in E. coli transposon 7. This plasmid was also shown to confer resistance to both streptomycin and spectinomycin by production of an adenylyltransferase that inactivated the drugs and the gene encoding this enzyme has also been sequenced. A second plasmid from the set, pCN2, was shown to inactivate streptomycin by a phosphotransferase mechanism and also to confer resistance to sulfonamides. The third plasmid from the set could not be correlated with a drug-resistance phenotype, but does appear to play a crucial role in plasmid mobilization.